PROJECT ABSTRACT Acute myeloid leukemia (AML) is a cancer of bone marrow-derived blood cells, where leukemic blasts build up and block proper function and development of myeloid progenitors. Conventional therapy eliminates most bulk tumor cells but disease-initiating leukemic stem cells (LSCs) survive, leading to disease progression and relapse2. Unlike bulk tumor cells and normal hematopoietic stem cells, LSCs rely on oxidative phosphorylation (OXPHOS). Thus, targeting OXPHOS is a promising strategy to selectively eradicate LSCs. The key metabolic drivers of OXPHOS in LSCs from relapsed patients are amino acid and fatty acid metabolism7. While we have previously described successful strategies for targeting amino acid metabolism8 the mechanisms that control fatty acid metabolism remain to be elucidated. Thus, the primary objective of this proposal is to better understand how fatty acids are metabolized to fuel OXPHOS in LSCs. LSCs in relapsed/refractory patients display increased fatty acid metabolism, which drives OXPHOS and LSC survival. We also show a strong correlation between fatty acid desaturase (FADS) expression and poor prognosis in AML. As unsaturated fatty acids are oxidized more rapidly than saturated9, increased FADS activity fuels OXPHOS even more than overall fatty acid metabolism. This suggests pharmacological targeting of fatty acid desaturation may offer a novel approach for LSC eradication in relapsed/refractory AML patients. We have also shown similar increases in fatty acid desaturation in cases of p53 loss in AML. Successful inhibition of OXPHOS is dependent on p53-driven apoptotic pathways, and p53 is a tight regulator of lipid metabolism. Therefore, a loss of p53 in AML may result in a loss of FADS inhibition and promotion of fatty acid desaturation. Increased unsaturated fatty acids may also drive inactivation of p53, resulting in further lipid aberrations. We hypothesize that relapsed/refractory LSCs upregulate fatty acid desaturation through increased FADS activity to maintain OXPHOS as a mechanism for survival. Our goal is to determine the mechanism by which relapsed/refractory LSCs maintain OXPHOS through fatty acid oxidation. Due to evidence linking loss of p53 and increased fatty acid desaturation, we also hypothesize that loss of p53 function in relapsed/refractory LSCs results in loss of inhibition of FADS1, increasing fatty acid desaturation. As increased unsaturated lipids modify p53 activity, this may drive continued p53 inactivation resulting in further lipid aberrations. These studies will determine whether inhibition of FADS1 prevents production of unsaturated fatty acids in relapsed/refractory LSCs, leading to novel therapeutic strategies. Together, the experiments described in this proposal will offer novel insights into the metabolism of relapsed/refractory LSCs and lay the groundwork for future clinical studies designed to better eradicate LSCs in AML patients.